The physiology of many organs in mammals is regulated by hormones. These hormones include steroid hormones, thyroid hormones, metabolites of vitamins, such as all trans retinoic acid, 9-cis retinoic acid, vitamin D and its metabolite 1,25 dihydroxyvitamin D3. These hormones are proteins and bind to intracellular receptors which regulate expression of genes.
There are a variety of receptors which respond to hormones. Osteoblasts and osteoclasts respond to steroid hormones, vitamin D and retinoic acid. Mammary epithelial cells and breast carcinoma cells respond to estrogens, progesterone, retinoic acid and glucocorticoids. Lymphocytes respond to glucocorticoids.
The response of receptors to hormones is particularly important in the development of a number of diseases, including cancer, osteoporosis and chronic inflammatory disease. For example, the vitamin D receptor is strongly implicated in the evolution of osteoporosis.
The hormone receptor family is called the nuclear hormone receptor family and consists not only of receptors whose ligands are known, but also of an increasing number of orphan receptors whose ligands are unknown.
The nuclear hormone receptors can be divided into several domains which include the hormone (ligand) binding domain, the DNA-binding domain and the transactivation domain. The DNA-binding domain consists of two zinc fingers and is responsible for the receptor""s binding to the DNA response elements which are found in the promoter and enhancer regions of the genes whose expression are regulated by these receptors. Once a hormone binds to its receptor, the receptor binds to the DNA thereby inducing gene transcription.
Proteins which modulate hormone receptor induced gene transcription are poorly understood. Such proteins are present in the nucleus of the cell and inhibit or promote the binding of a hormone to its receptor.
Calreticulin was initially identified as the major Ca2+-storage protein in the sarcoplasmic reticulum of skeletal muscle. Subsequent work has revealed that the protein can also be detected in the endoplasmic reticulum of non-muscle tissues. Calreticulin has been considered to be a resident protein of the endoplasmic reticulum of a cell, where it is thought to behave as a calcium binding protein due to its high capacity calcium binding properties. Calreticulin possesses many diverse functional domains such as high affinity, low capacity- and low affinity, high capacity-Ca2+-binding sites, a C-terminal KDEL endoplasmic reticulum retention signal, and a nuclear localization signal.
Calreticulin is also present in the nucleus of a cell, and it has been shown to have a consensus nuclear localization sequence which is highly homologous to that of histone proteins. Calreticulin is involved in DNA binding by nuclear hormone receptor and nuclear hormone receptor mediated gene transcription.
Calreticulin also plays a role in regulation of integrin activity. Calreticulin associates with the cytoplasmic domains of integrin subunits. Integrins are mediators of cell adhesion to extracellular ligands. They can transduce biochemical signals into and out of cells. The cytoplasmic domains of integrins interact with several structural and signalling proteins and participate in the regulation of cell shape, motility, growth and differentiation. The interaction between calreticulin and the cytoplasmic domains of integrin subunits can influence integrin-mediated cell adhesion to extracellular matrix.
To help design pharmaceuticals and therapies for certain diseases, one must understand the function of certain intracellular proteins, such as calreticulin, and their role in modulating hormone responsiveness. One must also understand the role of proteins, such as calreticulin, in integrin mediation of biochemical signals, extracellular calcium influx, cell adhesion and cell migration. Cells deficient in a protein can be used to study the physiological effects of the deficiency and identify substances to treat the deficiency. Substances may be also identified which activate or inhibit production of the protein or which affect the protein""s activity in cells not deficient in the protein. These substances can be used to inhibit or activate DNA binding by nuclear hormone receptor or nuclear hormone receptor induced gene transcription. They could inhibit or activate integrin mediation of biochemical signals, extracellular calcium influx, extracellular calcium influx, cell adhesion or cell migration. Pharmaceuticals including such peptides or their mimetics could be used to inhibit or activate DNA binding by nuclear hormone receptor or nuclear hormone receptor induced gene transcription. They could inhibit or activate integrin mediation of biochemical signals, extracellular calcium influx, cell adhesion or cell migration. Gene therapy could be used to inhibit or activate DNA binding by nuclear hormone receptor or nuclear hormone receptor induced gene transcription. It could also be used to inhibit or activate integrin mediation of biochemical signals, extracellular calcium influx, cell adhesion or cell migration.
A need exists to identify calreticulin-deficient cells which can be used as a research tool to identify and evaluate the physiological effects of calreticulin. These cells could be used to identify substances which can treat calreticulin deficiency. These cells could also be used to identify substances which activate or inhibit calreticulin production and activity. This would lead to improved methods of treating a variety of diseases, disorders and abnormal physical states in mammals by regulating hormone receptor induced gene transcription in mammalian cells.
The advent of gene targeting technology, sometimes referred to as xe2x80x9cgene knock-outxe2x80x9d, has allowed considerable insight into the role and function of particular gene products during development and differentiation. The technique relies on the use of pluripotent embryo-derived stem (ES) cells. An inactivating mutation is engineered into a cloned genomic fragment of the target gene and this mutated gene is introduced into ES cells cultured in vitro. Although the transfected mutant gene most frequently integrates randomly into the host cell""s genome, powerful selection schemes have been designed that allow the identification and isolation of the rare cells that have incorporated the mutant gene at the corresponding targeted chromosomal location through homologous recombination, thus creating a null allele of the target gene. These cells are then micro-injected into the blastocoel cavity of a preimplantation mouse embryo and the blastocyst is re-implanted into the uterus of a foster mother. Strains of mice with different coat colors are normally selected for the ES cell population and the recipient blastocyst, thus allowing simple identification of the chimeric animals on the basis of fur color. Back-crossing breeding then allows one to determine if the ES cells have contributed to the germ line of the chimeric animals. The progeny that shows ES cells germ line transmission is genotyped to detect the animals that carry the engineered mutation. These heterozygote siblings are then interbred to obtain animals that are homozygous for the desired mutation.
We have successfully targeted the 25-hydroxyvitamin D 24-hydroxylase (24-OHase) gene (St-Arnaud et al. Targeted inactivation of the 24-hydroxylase gene in embryonic stem (ES) cells. Journal of Bone and Mineral Research 9 (Suppl 1): S290 (1994)). Targeted ES cells were injected into mouse blastocysts by an outside facility, the MRC Centre of Excellence for Transgenesis. One of the resulting chimeric mice has transmitted the targeted allele to its progeny; animals that are heterozygous for the engineered mutation are normal and fertile. Animals homozygous for the targeted 24-OHase mutation are born with the expected Mendelian frequency of 25% (36/154); however, about one-half of the homozygotes died within one week after birth. We suspect that the incomplete penetrance of the homozygous phenotype may be due to the mixed genetic background (129Svxc3x97C57 BI 6) of the animals and are currently back-crossing the mutation into the inbred 129Sv background. Preliminary data suggest a mild hypercalcemia in homozygous mutants; histological examination revealed normal bone structure. Homozygous animals that survive are fertile. Interestingly, preliminary results suggest that bone development was abnormal in homozygotes born of homozygous females. These analyses should yield valuable insight into the biological role of 24,25(OH)2D3 in mineral homeostasis and bone development.
Calreticulin has been shown to regulate multiple functions in various cell types: calcium binding; cell attachment via its interaction with integrin receptors; and modulation of DNA binding by nuclear hormone receptor. Some of these roles of calreticulin may depend on strict stochiometric relationships between calreticulin and its target proteins. Inactivating one calreticulin allele alters this stoichiometry through a gene dosage effect and affect development of heterozygous mice. Since calreticulin has been shown to modulate the action of the AR, RAR and VDR, we concentrate on the morphology and function of some of the target tissues of these hormones: adrenal glands, gonads, lungs, and bone. Moreover, as cell attachment via the integrin receptors is compromised in heterozygous animals, they exhibit a higher incidence of carcinogenesis.
Calreticulin-deficient cells would greatly increase the ability to study the effects of calreticulin. These cells would allow identification of substances which affect calreticulin activity and production. These cells could also be used to identify substances which reverse the effects of calreticulin deficiency. There are currently no calreticulin-deficient cells available to researchers. There is a need for calreticulin-deficient cells which are stable. Ideally, cells that are homozygous and cells that are heterozygous for the calreticulin mutation would be isolated.